The use of microbial insecticide in agriculture can be part of a larger integrated pest management program. Bacillus thuringiensis (Bt) based biopesticide has been proven after decades of use as a safe alternative to chemical insecticides. The insecticidal crystal proteins produced by Bacillus thuringiensis are broadly used to control insect pests with agricultural importance. Bt proteins can be used in agriculture via microbial pesticides and genetically modified crop plants.
Bt is a spore-forming, Gram-positive bacterium, that can be isolated from many environments (Chaufaux et al., 1997; Martin and Travers, 1989) and new Bt strains have been isolated from soil (Carozzi et al., 1991a; DeLucca et al., 1979; Martin and Travers, 1989; Smith and Couche, 1991), leaves (Kaelin et al., 1994; Smith and Couche, 1991), and insects (Carozzi et al., 1991b) worldwide. Bt produces one or more delta-endotoxins or Cry proteins, which form insoluble inclusions known as insecticidal crystal proteins (ICPs). Although a specific Bt toxin has a narrow spectrum of activity, many different types of Bt toxins have been characterized that have selective toxicity to different orders of insects (Schnepf et al., 1998). Bt is also the main source of genes for transgenic expression in crops to provide pest control with few or no chemical pesticide applications. However, the narrow spectrum of activity for specific Bt toxins also limits efficacy, resulting in additional chemical pesticide applications for adequate pest control.
A generally accepted mode of action for Cry toxins describes the sequential steps of protoxin activation, specific-binding, and cell toxicity (Schnepf et al., 1998). Ingested ICPs are solubilized and activated to a toxic form by the insect's digestive fluids. After crossing the peritrophic matrix, activated toxins bind to specific proteins (i.e. cadherin and aminopeptidase-N) on the midgut microvilli. A recent model (Bravo et al., 2004) proposes that monomeric toxin binds a cad glycosylphosphatidylinositolherin, facilitating further processing necessary for toxin oligomerization. Toxin oligomers have high-affinity to proteins that are attached to the cell membrane by a (GPI) anchor, such as aminopeptidase or alkaline phosphatase. This binding and the localization of GPI-anchored proteins in specific membrane regions called lipid rafts result in toxin oligomer insertion, formation of pores or ion channels, and cell death by osmotic shock. An alternative model proposes the activation of intracellular signaling pathways by toxin monomer binding to cadherin without the need of the toxin oligomerization step to cause cell death (Zhang et al., 2005). Midgut lesions caused by the toxins led to septicemia induced by midgut bacteria that eventually leads to insect death (Broderick et al., 2006).
The cadherin Bt-R1 is a receptor for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. We also discovered that a peptide containing this region expressed in Escherichia coli functions as an enhancer of Cry1A toxicity against lepidopteran larvae (Chen et al., 2007).
US-2005-0283857-A1, U.S. Pat. No. 7,396,813, and WO 2005/07014A2 relate to the discovery and development of a Bt synergist that enhances Bt toxicity against insects that are agriculturally important pests. More specifically, these patent references relate to fragments of insect cadherins that can be used to enhance the toxicity of insecticidal crystal proteins produced by Bt some of which are commercial microbial biopesticides and some of which are expressed in transgenic plants. For ease of reference, we these peptides can be called “BtBoosters” or “BtB”. BtBooster can be mixed with commercial formulations of Bt to increase the value of the formulations. BtBooster can also be co-expressed with Bt toxin in Bt transgenic plants to offer better pest protection.
United States Patent Applications 2005010188439 (McCutcheon) relates to using a lipase polypeptide having insecticidal activity together with a Bt insecticidal protein.
The use of microbial insecticide in agriculture can be part of a larger integrated pest management program. Bacillus thuringiensis (Bt) based biopesticide has been proven after decades of use as a safe alternative to chemical insecticides. The insecticidal crystal proteins produced by Bacillus thuringiensis are broadly used to control insect pests with agricultural importance. Bt proteins can be used in agriculture via microbial pesticides and genetically modified crop plants.
Bt is a spore-forming, Gram-positive bacterium, that can be isolated from many environments (Chaufaux et al., 1997; Martin and Travers, 1989) and new Bt strains have been isolated from soil (Carozzi et al., 1991a; DeLucca et al., 1979; Martin and Travers, 1989; Smith. and Couche, 1991), leaves (Kaelin et al., 1994; Smith and Couche, 1991), and insects (Carozzi et al., 1991b) worldwide. Bt produces one or more delta-endotoxins or Cry proteins, which form insoluble inclusions known as insecticidal crystal proteins (ICPs). Although a specific Bt toxin has a narrow spectrum of activity, many different types of Bt toxins have been characterized that have selective toxicity to different orders of insects (Schnepf et al., 1998). Bt is also the main source of genes for transgenic expression in crops to provide pest control with few or no chemical pesticide applications. However, the narrow spectrum of activity for specific Bt toxins also limits efficacy, resulting in additional chemical pesticide applications for adequate pest control.
A generally accepted mode of action for Cry toxins describes the sequential steps of protoxin activation, specific-binding, and cell toxicity (Schnepf et al., 1998). Ingested ICPs are solubilized and activated to a toxic form by the insect's digestive fluids. After crossing the peritrophic matrix, activated toxins bind to specific proteins (i.e. cadherin and aminopeptidase-N) on the midgut microvilli. A recent model (Bravo et al., 2004) proposes that monomeric toxin binds a cad glycosylphosphatidylinositolherin, facilitating further processing necessary for toxin oligomerization. Toxin oligomers have high-affinity to proteins that are attached to the cell membrane by a (GPI) anchor, such as aminopeptidase or alkaline phosphatase. This binding and the localization of GPI-anchored proteins in specific membrane regions called lipid rafts result in toxin oligomer insertion, formation of pores or ion channels, and cell death by osmotic shock. An alternative model proposes the activation of intracellular signaling pathways by toxin monomer binding to cadherin without the need of the toxin oligomerization step to cause cell death (Zhang et al., 2005). Midgut lesions caused by the toxins led to septicemia induced by midgut bacteria that eventually leads to insect death (Broderick et al., 2006).
The cadherin Bt-R1 is a receptor for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. We also discovered that a peptide containing this region expressed in Escherichia coli functions as an enhancer of Cry1A toxicity against lepidopteran larvae (Chen et al., 2007).
US-2005-0283857-A1, U.S. Pat. No. 7,396,813, and WO 2005/07014A2 relate to the discovery and development of a Bt synergist that enhances Bt toxicity against insects that are agriculturally important pests. More specifically, these patent references relate to fragments of insect cadherins that can be used to enhance the toxicity of insecticidal crystal proteins produced by Bt some of which are commercial microbial biopesticides and some of which are expressed in transgenic plants. For ease of reference, these peptides can be called “BtBoosters” or “BtB”. BtBooster can be mixed with commercial formulations of Bt to increase the value of the formulations. BtBooster can also be co-expressed with Bt toxin in Bt transgenic plants to offer better pest protection.
United States Patent Applications 2005010188439 (McCutcheon) relates to using a lipase polypeptide having insecticidal activity together with a Bt insecticidal protein.
WO 03/018810 (by Syngenta) discusses some possibilities for adding Western corn rootworm (WCRW) cathepsin G favored sites (AAPF, AAPM, AVPF, PFLF) to B.t. Cry3A proteins.